This invention relates to a system and a method for generating electromagnetic fields in areas accessible to substances to be irradiated. This invention relates in particular to optical sensors for measuring biological or chemical substances.
Electromagnetic fields are utilized in numerous applications for detecting minute concentrations of substances most typically in liquid samples. There are two basic categories of optical sensors designed to detect substance accumulations on the detector surface: Those used for labeled substances (label methodology) and those that function without such labeling (non-label methodology).
Label methodology encompasses, inter alia, a method whereby the substances to be detected are labeled with a fluorescent dye. Examples of commercially available fluorescent dyes include CY5 for an excitation wavelength of 532 nm, and CY3 for an excitation wavelength of around 635 nm.
While such labeling may be selective, it usually also covers other substances that are present in a sample to be measured. In cases of nonselective labeling, selectivity can be achieved for instance by applying on the surface of the sensor a treatment that allows only the labeled substance that is to be measured to selectively adhere to that surface. When, after full adhesion, the surface is flushed, the intensity of the resultant fluorescence signal provides a quantitative indication of the concentration of the substance to be measured in the sample concerned. Such quantitative information can also be obtained without flushing whenever it is possible to cause a fluorescence-stimulating electromagnetic field to attach itself in concentrated and essentially dedicated fashion to the substances that are to be detected on the sensor surface.
As long as not all of the labeled substances adhering to the surface fluoresce, this general rule applies: the higher the electromagnetic field intensity generated on the surface relative to the impinging light intensity, the better the signal-to-noise (S/N) ratio of the measurement. This has a direct effect on the measuring sensitivity of the sensor. Accordingly, one will try to produce on the surface area concerned as strong an electromagnetic field as possible in relation to the volume to be measured.
Apart from the label-based methods described, i.e. marker-based measuring techniques, non-label methods are widely used as well. In that case, it is for instance a change of the angle of refraction caused by the accumulation of the substance to be measured on the sensor surface that is a factor directly influencing the field distribution in the sensor. That factor has an effect on such optical measuring parameters as the diffraction coefficient or perhaps the waves that pass through a fiber-optic cable, triggering mensurable changes. Here as well it is important that at least segments of the substances being measured have areas accessible to electromagnetic fields.
One possibility to build up that kind of field distribution accessible to the substance being measured is to utilize cross-attenuated electromagnetic waves. In the simplest case a one-time total internal reflection (TIR) is used. To that end, excitation light emanating from the substrate impinges on the sensor surface at an angle that is greater than the critical total reflection angle. This generates on the surface a cross-attenuated field which, declining exponentially, projects from the substrate into the medium to be measured. Technical literature also refers to these fields as evanescent fields, since no light propagates into the medium to be measured but, instead, the field only “projects” into the medium and is thus restricted to the immediate contact surface within the corresponding boundaries.
The so-called depth of penetration, meaning the distance from the contact surface at which the field intensity has dropped to 1/e (where e is the Eulerian coefficient), depends among other factors on the actual angle of incidence and is typically measured in units of the vacuum wavelength of the excitation energy while being of the same order of magnitude.
One way to elevate this type of evanescent field relative to simple total reflection is to cause the excitation light energy to reflect off the contact surface multiple times. For example, if an optical layer of a specific thickness and with a high refractive index relative to the substrate is applied on the latter, it is possible, under certain conditions, for total reflection to take place on both contact surfaces of the layer and for the light propagation to be guided within the layer in a so-called waveguide mode. In a suitably selected waveguide configuration, this leads to an elevated evanescent field on the surface of the waveguide.
It is important in this context that the wave-conducting layer, meaning the layer in which the light wave is guided, have a refractive index that is greater than the refractive indices of the substrate and of the medium next to the waveguide layer. Otherwise there cannot be multiple total reflections nor, consequently, any wave propagation (refer for instance to WO 86/071149). For a given substrate the choice of materials for the wave-conducting layer is therefore limited to high-refraction materials.
Another problem is posed by the fact that the mode propagation and thus the intensity of the field that is available for excitation depends in highly sensitive fashion on the waveguide configuration and possibly existing imperfections. Even minute impurities cause light scattering and a diminution of the intensity of the light passing through the waveguide. That effect is integrally propagated over the entire distance of the light path through the waveguide and even in the case of minute impurities and/or defects it can produce incorrect measurements.
Another difficulty is encountered when coupling the light into the waveguide, which can be accomplished through end-face interfacing, prismatic coupling or via a coupler grating. In all of these it is difficult to ensure constancy in terms of coupling efficiency which, however, should preferably be obtainable for quantitative measurements.
As another problem, the density of the various measuring ranges is limited due to the expansion of the waveguide.
Another way to arrive at an elevated evanescent field is to stimulate so-called surface plasmons. These excitation conditions, generated in metallic layers, propagate in the plane of the layer until they fall apart for instance by absorption in the metal or by scattering. Here again, controlling the evanescent field intensity produced by surface plasmons is quite difficult. And again, there is a limit to the density of the measuring ranges since surface plasmons usually travel over finite distances only before they decay. Moreover, in many cases, metals have stability problems and most of all they tend to age, potentially leading to unreliable measurements.
More recent approaches utilize the well-known effect of the resonant grating with anomalous transmission breaks, for instance as described by Novartis in WO2001/002839 (hereinafter referred to as the Novartis application). As in the case of the waveguide, the substrate is coated with a layer whose refractive index is higher than the refractive index of the substrate, since otherwise there would be no resonance effect. In addition, the surface area of the measuring field is defined by a periodic pattern of channels.
The dimensions of the structures and layers are so chosen that the impingement of coherent light at a particular angle causes a resonance effect whereby transmission is reduced in anomalous fashion, building up the desired evanescent field. The advantage of that approach is that it is not necessary for the light to travel waveguide-style over a long distance, making the system substantially less sensitive to imperfections and centers of scattering. Moreover, compared to waveguide coupling provisions the system can be smaller in size, allowing for a considerably larger number of measuring ranges since for all practical purposes the light does not propagate laterally. This advantage of a potentially greater density of measuring ranges has been stressed most of all in WO2000/75644 by Zeptosens (hereinafter referred to as the Zeptosens application). The Zeptosens application as well provides for continuous modulations in the measuring range, although it is still based on a laminar optical waveguide.
Both the waveguide approach for generating a high field intensity in the surface area and the resonant-grating approach employing anomalous transmission suffer from a drawback in that the field intensity obtained on the surface is a mere fraction of the field intensity present in the highly refractive layer and perhaps in the wave-conducting layer. In these systems, only the cross-attenuated evanescent spurs of the field are accessible.
Another disadvantage lies in the fact that in the cases concerned a highly refractive layer must serve as the terminal surface relative to the surrounding medium. The most progress here has been made in biochemical applications on SiO2 surfaces. With glass substrates, other materials must be used for producing a layer which compared to the substrate has a high refractive index. Examples of the materials employed include TiO2 or Ta2O5. Compared to SiO2, however, these materials have been less widely adopted for biochemical applications. It is possible to coat the high-refraction layer with a thin film of SiO2, which in fact must be very thin since the decline of the evanescent field is exponential in this film as well.